Field of the Invention
The present invention relates to phenylacetamido-pyrazole derivatives, to a process for their preparation, to pharmaceutical compositions containing them, and to their use as therapeutic agents, particularly in the treatment of cancer and cell proliferation disorders.
Discussion of the Background
Several cytotoxic drugs such as, e.g., fluorouracil (5-FU), doxorubicin and camptothecins damage DNA or affect cellular metabolic pathways and thus cause, in many cases, an indirect block of the cell cycle. Therefore, by producing an irreversible damage to both normal and tumor cells, these agents result in a significant toxicity and side effects.
In this respect, compounds capable of functioning as highly specific antitumor agents by selectively leading to tumor cell arrest and apoptosis, with comparable efficacy but reduced toxicity than the currently available drugs, are desirable.
It is well known that progression through the cell cycle is governed by a series of checkpoint controls, otherwise referred to as restriction points, which are regulated by a family of enzymes known as the cyclin-dependent kinases (cdk). In turn, the cdks themselves are regulated at many levels such as, for instance, binding to cyclins.
The co-ordinated activation and inactivation of different cyclin/cdk complexes is necessary for normal progression through the cell cycle. Both the critical G1-S and G2-M transitions are controlled by the activation of different cyclin/cdk activities. In G1, both cyclin D/cdk4 and cyclin E/cdk2 are thought to mediate the onset of S-phase. Progression through S-phase requires the activity of cyclin A/cdk2 whereas the activation of cyclin A/cdc2 (cdk1) and cyclin B/cdc2 are required for the onset of mitosis. For a general reference to cyclins and cyclin-dependent kinases see, for instance, Kevin R. Webster et al, in Exp. Opin. Invest. Drugs, 1998, Vol. 7(6), 865-887.
Checkpoint controls are defective in tumor cells due, in part, to disregulation of cdk activity. For example, altered expression of cyclin E and cdks has been observed in tumor cells, and deletion of the cdk inhibitor p27 KIP gene in mice has been shown to result in a higher incidence of cancer.
Increasing evidence supports the idea that the cdks are rate-limiting enzymes in cell cycle progression and, as such, represent molecular targets for therapeutic intervention. In particular, the direct inhibition of cdk/cyclin kinase activity should be helpful in restricting the unregulated proliferation of a tumor cell.
It is an object of the invention to provide compounds that are useful in treating cell proliferative disorders associated with an altered cell cycle dependent kinase activity. It is another object to provide compounds that have cdk/cyclin kinase inhibitory activity.
The present inventors have now discovered that certain phenylacetamido-pyrazoles are endowed with cdk/cyclin kinase inhibitory activity and are thus useful in therapy as antitumor agents and lack, in terms of both toxicity and side effects, the aforementioned drawbacks associated with currently available antitumor drugs.
More specifically, the phenylacetamido-pyrazoles of the invention are useful in the treatment of a variety of cancers including, but not limited to: carcinoma such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage including leukaemia, acute lymphocitic leukaemia, acute lymphoblastic leukaemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin""s lymphoma, non-Hodgkin""s lymphoma, hairy cell lymphoma and Burkett""s lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemtas, myelodysplastic syndrome and promyelocytic leukaemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma neuroblastoma, glioma and schwannomas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratocanthoma, thyroid follicular cancer and Kaposi""s sarcoma.
Due to the key role of cdks in the regulation of cellular proliferation, these phenylacetamido-pyrazole derivatives are also useful in the treatment of a variety of cell proliferative disorders such as, for example, benign prostate hyperplasia, familial adenomatosis polyposis, neurofibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-surgical stenosis and restenosis.
The compounds of the invention may be useful in treatment of Alzheimer""s disease, as suggested by the fact that cdk5 is involved in the phosphorylation of tau protein (J. Biochem. 117, 741-749, 1995).
The compounds of this invention, as modulators of apoptosis, may also be useful in the treatment of cancer, viral infections, prevention of AIDS development in HIV-infected individuals, autoimmune diseases and neurodegenerative disorders.
The compounds of this invention may be useful in inhibiting tumor angiogenesis and metastasis.
The compounds of the invention may also act as inhibitor of other protein kinases, e.g., protein kinase C in different isoforms, Met, PAK-4, PAK-5, ZC-1, STLK-2, DDR-2, Aurora 1, Aurora 2, Bub-1, PLK, Chk1, Chk2, HER2, raf1, MEK1, MAPK, EGF-R, PDGF-R, FGF-R, IGF-R, P13K, weel kinase, Src, Abl, Akt, MAPK, ILK, MK-2, IKK-2, Cdc7, Nek, and thus be effective in the treatment of diseases associated with other protein kinases.
The compounds of the invention are also useful in the treatment and prevention of radiotherapy-induced or chemotherapy-induced alopecia.
Accordingly, the present invention provides a method for treating cell proliferative disorders associated with an altered cell cycle dependent kinase activity, by administering to a mammal in need thereof an effective amount of a phenylacetamido-pyrazole derivative represented by formula (I): 
wherein
R is a C3-C5 cycloalkyl group;
R1 is a hydrogen atom or a methyl group;
or a pharmaceutically acceptable salt thereof.
In a preferred embodiment of the method described above, the cell proliferative disorder is selected from the group consisting of cancer, Alzheimer""s disease, viral infections, autoimmune diseases and neurodegenerative disorders.
Specific types of cancer that may be treated include carcinoma, squamous cell carcinoma, hematopoietic tumors of myeloid or lymphoid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratocanthoma, thyroid follicular cancer, and Kaposi""s sarcoma.
In another preferred embodiment of the method described above, the cell proliferative disorder is selected from the group consisting of benign prostate hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-surgical stenosis and restenosis.
In addition, the inventive method provides tumor angiogenesis and metastasis inhibition. The inventive method may also provide cell cycle inhibition or cdk/cyclin dependent inhibition.
In addition to the above, the method object of the present invention provides treatment and prevention of radiotherapy-induced or chemotherapy-induced alopecia.
The present invention also provides a phenylacetamido-pyrazole derivative represented by formula (I): 
wherein
R is a C3-C5 cycloalkyl group;
R1 is a hydrogen atom or a methyl group;
or a pharmaceutically acceptable salt thereof.
The present invention also includes methods of synthesising the phenylacetamido-pyrazole derivatives represented by formula (I). A pharmaceutical composition comprising the phenylacetamido-pyrazole derivatives represented by formula (I) is also included in the present invention.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description.
Several carbonylamino-pyrazole derivatives are known in the art, for instance as pesticides, herbicides or even as therapeutic agents. Among these are, as an example, heteroaryl-pyrazoles active as p38 kinase inhibitors (WO 98/52941, G. D. Searle and Co.) and 3-amino-pyrazoles active as protein kinase inhibitors (WO 96/14843, COR Therapeutics, Inc.).
The international patent application WO 01/12189 in the name of Pharmacia and Upjohn S.p.A. and Pharmacia and Upjohn Co., herewith incorporated by reference, discloses a class of carbonylamino-pyrazoles further substituted by cycloalkyl groups within the pyrazole ring, which possess cell cycle dependent kinase inhibitory activity.
The compounds of the present invention fall within the scope of the general formula of WO 01/12189 but are not specifically exemplified therein.
In addition, a class of carbonylamino-pyrazoles endowed with cyclin dependent kinase inhibitory activity is also disclosed in the US provisional patent application No. 60/252911 filed on Nov. 27, 2000 in the name of Pharmacia and Upjohn S.p.A., which is herewith incorporated by reference.
As it will be readily appreciated, the unsubstituted ring nitrogen pyrazoles in the compounds of the invention are known to rapidly equilibrate, in solution, as admixtures of both tautomers: 
Accordingly, in the present invention and unless specifically noted otherwise, where only one tautomer is indicated for the compounds of formula (I), the other (Ia), is also within the scope of the present invention.
The compounds of formula (I) may have asymmetric carbon atoms and may therefore exist either as racemic admixtures or as individual optical isomers which are all within the scope of the present invention.
As an example, the compounds of formula (I) wherein R1 is methyl have an asymmetric carbon atom and, hence, both the (R) and (S) optical isomers as well as the racemic (R,S) admixture are within the scope of the invention.
Likewise, the use as an antitumor agent of all the possible isomers and their admixtures and of both the metabolites and the pharmaceutically acceptable bio-precursors (otherwise referred to as pro-drugs) of the compounds of formula (I) are also within the scope of the present invention.
As used herein, unless otherwise specified, the term C3-C5 cycloalkyl comprises cyclopropyl, cyclobutyl and cyclopentyl.
Because of possible protonation reactions at a pyrazole nitrogen atom, the compounds of the invention may give rise to acid addition salts.
Pharmaceutically acceptable salts of the compounds of formula (I) include the acid addition salts with inorganic or organic acids such as, for instance, nitric, hydrochloric, hydrobromic, sulphuric, perchloric, phosphoric, acetic, trifluoroacetic, propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulphonic, isethionic and salicylic acid.
Preferred compounds of the invention are the compounds of formula (I) wherein R is cyclopropyl.
Another class of preferred compounds of the invention are those wherein R1 is a methyl group.
Still more preferred are the compounds wherein both features are combined so as to get a compound of formula (I) wherein R is cyclopropyl and R1 is methyl.
Even more preferred is the compound of formula (I) wherein R is cyclopropyl and R1 is methyl, in its (S) optical form, namely (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide.
Examples of compounds of formula (I) of the invention, optionally in the form of pharmaceutically acceptable salts, are:
1. N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
2. (2R)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
3. (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
4. N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]acetamide;
5. N-(5-cyclobutyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
6. (2R)-N-(5-cyclobutyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
7. (2S)-N-(5-cyclobutyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
8. N-(5-cyclobutyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]acetamide;
9. N-(5-cyclopentyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
10. (2R)-N-(5-cyclopentyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
11. (2S)-N-(5-cyclopenyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide;
12. N-(5-cyclopentyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]acetamide;
The compounds of formula (I) and the pharmaceutically acceptable salts thereof, object of the invention, may be obtained by a process comprising:
a) reacting the compounds of formula (II) or the regioisomers of formula (IIa) 
xe2x80x83wherein R is as above defined and P represents a suitable nitrogen-pyrazole protecting group, with the compounds of formula (III) 
xe2x80x83wherein R1 is as above defined and Rxe2x80x2 represents hydroxy or a halogen atom, thus obtaining the compounds of formula (IV) or (IVa), respectively 
b) and deprotecting the compounds of formula (IV) or (IVa) so as to obtain the derivatives of formula (I) and, if desired, converting them into pharmaceutically acceptable salts thereof.
The above process is an analogy process which can be carried out according to well known methods.
According to step a) of the process, the reaction of the compounds of formula (II) or (IIa) with the compounds of formula (III) can be carried out in the presence of a coupling agent, for instance a carbodiimide such as 1,3-dicyclohexylcarbodiimide, 1,3-diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, optionally in the presence of a tertiary base such as triethylamine, N-methylmorpholine, N,N-diisopropylethylamine or pyridine.
The reaction occurs in a suitable solvent such as, for example, dichloromethane, chloroform, tetrahydrofuran, diethylether, 1,4-dioxane, acetonitrile, toluene or N,N-dimethylformamide, at a temperature ranging from about xe2x88x9210xc2x0 C. to reflux and for a suitable time, for instance from about 30 minutes to about 96 hours.
Alternatively, the reaction of the compounds of formula (II) or (IIa) with the compounds of formula (III) can be also carried out by a mixed anhydride method, that is by using an alkyl chloroformate such as ethyl, isobutyl or isopropylchloroformate in the presence of a tertiary base such as triethylamine, N-methylmorpholine, N,N-diisopropylethylamine or pyridine, in a suitable solvent such as toluene, dichloromethane, chloroform, tetrahydrofuran, acetonitrile, diethylether, 1,4-dioxane or N,N-dimethylformamide, at a temperature ranging from about xe2x88x9230xc2x0 C. to room temperature.
When starting from the compounds of formula (III) wherein Rxe2x80x2 is a halogen atom, for instance chlorine, the reaction can be carried out in the presence of a base such as triethylamine, N-methylmorpholine, N,N-diisopropylethylamine or pyridine, in a suitable solvent such as ethyl acetate, dichloromethane, chloroform, diethylether, tetrahydrofuran, 1,4-dioxane, acetonitrile, toluene or N,N-dimethylformamide, at a temperature ranging from about 0xc2x0 C. to reflux.
As far as the compounds of formula (II) or (IIa) are concerned, suitable P groups are those conventionally used to protect pyrazole-nitrogen atoms.
Preferably, for both compounds (II) and (IIa), P represents a tert-butoxycarbonyl (BOC) group.
In step b) of the process, the compounds of formula (IV) or (IVa) are converted into the desired derivatives of formula (I) by deprotecting the pyrazole-nitrogen atom, according to conventional methods. As an example, deprotection from BOC may occur by acidic hydrolysis, for instance in the presence of trifluoroacetic, formic or sulphuric acid, in a suitable solvent such as dichloromethane, 1,4-dioxane or ethanol, at a temperature ranging from about 0xc2x0 C. to room temperature.
The optional salification of a compound of formula (I) or, alternatively, the conversion of a salt thereof into the free compound, can be all carried out by conventional methods.
The compounds of formula (II) or (IIa) are known compounds or may be prepared according to known methods by starting from the corresponding deprotected amino-pyrazoles.
For a reference to the preparation of the compounds of formula (II) see, for instance, the aforementioned WO 01/12189.
When preparing the compounds of formula (IIa), the R-substituted amino-pyrazoles are protected, for instance as BOC derivatives, through reaction with tert-butoxycarbonylanhydride in the presence of a dichloromethane/water admixture and of an inorganic base such as sodium hydroxide, carbonate or hydrogenocarbonate. Alternatively, this same reaction may be also carried out in dichloromethane, chloroform, toluene, tetrahydrofuran or 1,4-dioxane, and in the presence of an organic base, for instance triethylamine or N,N-diisopropylethylamine, at a temperature ranging from about 0xc2x0 C. to room temperature.
Alternatively, the compounds of formula (IIa), for instance protected as BOC derivatives, can be prepared by reacting the corresponding keto-nitriles of formula (V) 
wherein R is as described above, with tert-butylcarbazate in a suitable solvent such as ethanol or methanol, in the presence of a base such as triethylamine or N,N-diisopropylethylamine, at a temperature ranging from about 0xc2x0 C. to room temperature.
The compounds of formula (III) wherein Rxe2x80x2 is a halogen atom, for instance chlorine, can be prepared from the corresponding derivatives of formula (III) wherein Rxe2x80x2 is hydroxy, by reacting these latter with oxalyl chloride or thionyl chloride, according to conventional methods for preparing acyl halides. The reaction may occur in the presence of a suitable solvent, for instance dichloromethane, tetrahydrofuran, ethyl acetate or toluene, at a temperature ranging from room temperature to reflux.
In their turn, the compounds of formula (III) wherein Rxe2x80x2 is hydroxy can be prepared by a process comprising reacting a derivative of formula (VI) 
wherein R1 is as defined above, with 2-chloroethylchloroformate, followed by cyclization in a basic medium.
In particular, the reaction with 2-chloroethylchloroformate can be carried out in the presence of aqueous sodium hydroxide and toluene at room temperature, or in a suitable solvent such as dichloromethane, chloroform or tetrahydrofuran with a conventional base under anhydrous conditions, at a temperature ranging from about xe2x88x9220xc2x0 C. to room temperature. The intermediate compounds of formula (VII) thus prepared 
wherein R1 is as above defined, is then treated with an aqueous base, for instance sodium hydroxide, in a suitable solvent such as water or water-methanol admixtures, at a temperature ranging from room temperature to reflux or, alternatively, with an inorganic or an organic base such as potassium carbonate or 1,8-diazabicyclo[5.4.0]undec-7-ene in N,N-dimethylformamide or N,N-dimethylacetamide at room temperature.
The compounds of formula (VI) are known or can be easily prepared according to known methods. When they are enatiomerically pure, they are intended as being obtained by resolution of the racemate, according to conventional methods.
Alternatively, the compounds of formula (I) and the pharmaceutically acceptable salts thereof can be prepared by a process comprising:
a) reacting the compounds of formula (VIII) 
xe2x80x83wherein R and R1 are as described above, with chloroethylchloroformate in the presence of a base such as pyridine, triethylamine or N,N-diisopropylethylamine and in a suitable solvent such as pyridine, dichloromethane, tetrahydrofuran or N,Nxe2x80x2-dimethylformamide, at a temperature ranging from about xe2x88x9220xc2x0 C. to room temperature, thus obtaining the compounds of formula (IX) 
xe2x80x83wherein R and R1 are as described above;
b) reacting the compounds of formula (IX) with a suitable base such as potassium carbonate or 1,8-diazabicyclo[5.4.0]undec-7-ene in a suitable solvent such as N,N-dimethylformamide or N,N-dimethylacetamide, at a temperature ranging from room temperature to reflux, thus obtaining the compounds of formula (X) 
c) and by hydrolyzing the compounds of formula (X) with a base such as triethylamine or potassium carbonate in a suitable solvent such as methanol.
In their turn, the compounds of formula (VIII) can be prepared by a process comprising:
a) reacting the compounds of formula (XI) 
xe2x80x83wherein R1 is as described above, with the compounds of formula (IIa) wherein R is as described above, thus obtaining the compounds of formula (XII) 
b) and by hydrolyzing the compounds of formula (XII) in acidic medium.
The reaction between the compounds of formula (XI) and the compounds of formula (IIa) can be carried out in the presence of a conventional organic base such as triethylamine, N-methylmorpholine or N,N-diisopropylethylamine, in a suitable solvent such as chloroform, dichloromethane, tetrahydrofuran or 1,4-dioxane, at a temperature ranging from about 0xc2x0 C. to room temperature.
The hydrolysis of the compounds of formula (XII) to afford the compounds of formula (VIII) can be carried out in a suitable solvent such as dichloromethane or ethanol with a suitable acid such as trifluoroacetic, formic or sulphuric acid at room temperature.
The compounds of formula (XI) can be prepared by a process comprising:
a) reacting the compounds of formula (VI) wherein R1 is as described above with tert-butoxycarbonylanhydride, thus obtaining the compounds of formula (XIII) wherein R1 is as described above 
b) and by reacting the compounds of formula (XIII) with oxalyl or thionyl chloride.
The reaction between the compounds of formula (VI) with tertbutoxycarbonylanhydride can be carried out in a suitable solvent such as admixtures water/1,4-dioxane or water/dichloromethane admixtures, in the presence of a base such as sodium carbonate or sodium hydroxide, at a temperature ranging from about 0xc2x0 C. to room temperature.
The reaction between the compounds of formula (XIII) with oxalyl or thionyl chloride can be carried out in a suitable solvent such as dichloromethane, tetrahydrofuran or ethyl acetate, in the presence of a catalytic amount of dimethylformamide at a temperature ranging from about 0xc2x0 C. to reflux.
As it will be readily appreciated, if the compounds of formula (I), prepared according to the processes described above, are obtained as an admixture of isomers, their separation into the single isomers of formula (I), according to conventional techniques, is within the scope of the present invention.
Conventional techniques for resolution of the racemate include, for instance, partitioned crystallization of diastereoisomeric salt derivatives or preparative chiral HPLC.
Pharmacology
The compounds of formula (I) are active as cdk/cyclin inhibitors as they gave positive results when tested according to the following procedure.
The inhibiting activity of putative cdk/cyclin inhibitors and the potency of selected compounds was determined through a method of assay based on the use of the MultiScreen-PH 96 well plate (Millipore), in which phosphocellulose filter paper was placed at each well bottom allowing binding of positive charged substrate after a washing/filtration step.
When a radioactivity labelled phosphate moiety was transferred by the ser/threo kinase to the filter-bound histone, light emitted was measured in a scintillation counter.
The inhibition assay of cdk2/Cyclin A activity was performed according to the following protocol.
Kinase reaction: 1.5 xcexcM histone H1 substrate, 25 xcexcM ATP (0.5 uCi P33xcex3-ATP), 100 ng Cyclin A/cdk2 complex, 10 xcexcM inhibitor in a final volume of 100 xcexcl buffer (TRIS HCl 10 mM pH 7.5, MgCl2 10 mM, 7.5 mM DTT) were added to each well of a 96 U bottom well plate. After 10 minutes at 37xc2x0 C. incubation, reaction was stopped by 20 xcexcl EDTA 120 mM.
Capture: 100 xcexcl were transferred from each well MultiScreen plate, to allow substrate binding phosphocellulose filter. Plates were then washed 3 times with 150 xcexcl/well PBS Ca++/Mg++ free and filtered by MultiScreen filtration system.
Detection: filters were allowed to dry at 37xc2x0 C., then 100 xcexcl/well scintillant were added and 33P labelled histone H1 was detected by radioactivity counting in the Top-Count instrument.
Results: data were analysed and expressed as % inhibition referred to total activity of enzyme (=100%).
All compounds showing inhibition  greater than 50% were further analysed in order to study and define the kinetic-profile of the inhibitor via Ki calculation.
The protocol used was the same described above, except for ATP and substrate concentrations. Either the concentrate of ATP and histone H1 substrate were varied: 4, 8, 12, 24, 48 xcexcM for ATP (containing proportionally diluted P33xcex3-ATP) and 0.4, 0.8, 1.2, 2.4, 4.8 xcexcM for histone were used in absence and presence of two different, properly chosen inhibitor concentrations.
Experimental data were analysed by the computer program xe2x80x9cSigmaPlotxe2x80x9d for Ki determination, using a random bireactant system equation:   v  =            Vmax      ⁢              xe2x80x83            ⁢                                    (            A            )                    ⁢                      xe2x80x83                    ⁢                      (            B            )                                                aK            A                    ⁢                      K            B                                      1      +                        (          A          )                          K          A                    +                                    xe2x80x83                    ⁢                      (            B            )                                    K          B                    +                                    (            A            )                    ⁢                      xe2x80x83                    ⁢                      (            B            )                                                aK            A                    ⁢                      K            B                              
where A=ATP and B=histone H1.
In addition, the inhibiting activity of putative cdk/cyclin inhibitors and the potency of selected compounds was determined using a method of assay based on the use of a SPA (Scintillation Proximity Assay) 96 well plate assay. The assay is based on the ability of streptavidin-coated SPA beads to capture a biotinylated peptide derived from a phosphorylation site of histone.
When a radioactivity labelled phosphate moiety was transferred by the serine/threonine kinase to the biotinylated histone peptide, light emitted was measured in scintillation counter.
The inhibition assay of cdk5/p25 activity was performed according to the following protocol.
Kinase reaction: 1.0 xcexcM biotinylated histone peptide substrate, 0.25 uCi P33xcex3-ATP, 4 nM cdk2/p25 complex, 0-100 xcexcM] inhibitor in a final volume of 100 xcexcl buffer (Hepes 20 mM pH 7.5, MgCl2 15 mM, 1 mM DTT) were added to each well of a 96 U bottom well plate. After 20 min at 37xc2x0 C. incubation, the reaction was stopped by the addition of 500 ug SPA beads in phosphate-buffered saline containing 0.1% Triton X-100, 50 M ATP and 5 mM EDTA. The beads were allowed to settle, and the radioactivity incorporated in the 33P-labelled peptide was detected in a Top Count scintillation counter.
Results: Data were analysed and expressed as % inhibition using the formula:
xe2x80x83100xc3x97(1xe2x88x92(Unknownxe2x88x92Bkgd)/(Enz. Controlxe2x88x92Bkgd))
IC50 values were calculated using a variation of the four parameter logistics equation:
Y=100/[1+10{circumflex over ( )}{(LogEC50xe2x88x92X)*Slope}]
Where X=log(uM) and Y=% Inhibition.
Given the above inhibition assays, the compounds of formula (I) of the invention resulted to possess a remarkable cdk inhibitory activity and are thus useful in therapy against proliferative disorders associated with an altered cell cycle dependent kinase activity.
In particular, when tested against cdk2/A, the representative compound of the invention (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]propanamide showed an inhibitory activity, expressed as IC50, of 8 nM.
By restricting the unregulated proliferation of tumor cells, the compounds of formula (I) are therefore useful in therapy in the treatment of various tumors such as, for instance, carcinomas, e.g., mammary carcinoma, bladder carcinoma, colon carcinoma, ovary endometrial tumors, sarcomas, e.g., soft tissue and bone sarcomas, and the hematological malignancies such as, e.g., leukemias.
In addition, the compounds of formula (I) are also useful in the treatment of other cell proliferative disorders such as psoriasis, vascular smooth cell proliferation associated with atherosclerosis and post-surgical stenosis and restenosis, and in the treatment of Alzheimer""s disease.
The compounds of the present invention can be administered either as single agents or, alternatively, in combination with known anticancer treatments such as radiation therapy or chemotherapy regimen in combination with cytostatic or cytotoxic agents, antibiotic-type agents, alkylating agents, antimetabolite agents, hormonal agents, immunological agents, interferon-type agents, cyclooxygenase inhibitors (e.g. COX-2 inhibitors), matrixmetalloprotease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti-angiogenesis agents (e.g. angiogenesis inhibitors), farnesyl transferase inhibitors, ras-raf signal transduction pathway inhibitors, cell cycle inhibitors, other cdks inhibitors, tubulin binding agents, topoisomerase I inhibitors, topoisomerase II inhibitors, and the like.
As an example, the compounds of the invention can be administered in combination with one or more chemotherapeutic agents such as, for instance, exemestane, formestane, anastrozole, letrozole, fadrozole, taxane and derivatives such as paclitaxel or docetaxel, encapsulated taxanes, CPT-11, camptothecin derivatives, anthracycline glycosides, e.g., doxorubicin, idarubicin, epirubicin, etoposide, navelbine, vinblastine, carboplatin, cisplatin, estramustine, celecoxib, tamoxifen, raloxifen, Sugen SU-5416, Sugen SU-6668, Herceptin, and the like, optionally within liposomal formulations thereof.
If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent within the approved dosage range.
Compounds of formula (I) may be used sequentially with known anticancer agents when a combination formulation is inappropriate.
The compounds of formula (I) of the present invention, suitable for administration to a mammal, e.g., to humans, can be administered by the usual routes and the dosage level depends upon the age, weight, conditions of the patient and administration route.
For example, a suitable dosage adopted for oral administration of a compound of formula (I) may range from about 10 to about 500 mg per dose, from 1 to 5 times daily. The compounds of the invention can be administered in a variety of dosage forms, e.g., orally, in the form tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form suppositories; parenterally, e.g., intramuscularly, or through intravenous and/or intrathecal and/or intraspinal injection or infusion.
The present invention also includes pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable excipient, which may be a carrier or a diluent.
The pharmaceutical compositions containing the compounds of the invention are usually prepared following conventional methods and are administered in a suitable pharmaceutical form.
For example, the solid oral forms may contain, together with the active compound, diluents, e.g., lactose, dextrose saccharose, sucrose, cellulose, corn starch or potato starch; lubricants, e.g., silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents, e.g., starches, arabic gum, gelatine methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disintegrating agents, e.g., starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. These pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
The liquid dispersions for oral administration may be, e.g., syrups, emulsions and suspensions.
As an example, the syrups may contain, as carrier, saccharose or saccharose with glycerine and/or mannitol and sorbitol.
The suspensions and the emulsions may contain, as examples of carriers, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g., sterile water, olive oil, ethyl oleate, glycols, e.g., propylene glycol and, if desired, a suitable amount of lidocaine hydrochloride.
The solutions for intravenous injections or infusions may contain, as a carrier, sterile water or preferably they may be in the form of sterile, aqueous, isotonic, saline solutions or they may contain propylene glycol as a carrier.
The suppositories may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g., cocoa butter, polyethylene glycol, a polyoxyethylene sorbitan fatty acid ester surfactant or lecithin.